Proc. Natl. Acad. Sci. USA Vol. 95, pp. 11246–11250, September 1998 Cell Biology

Very late DNA replication in the human cell cycle

ʈ R. J. WIDROW*†,R.SCOTT HANSEN‡,HIROSHI KAWAME‡,STANLEY M. GARTLER‡§, AND CHARLES D. LAIRD†§¶

*Molecular and Cellular Biology Program of the University of Washington and Fred Hutchinson Cancer Research Center, Fred Hutchinson Cancer Research Center, Seattle, WA 98104; Departments of ‡Medicine, §Genetics, and ¶Zoology, University of Washington, Seattle, WA 98195; and ʈMolecular Medicine Division, C3-168, Fred Hutchinson Cancer Research Center, Seattle, WA 98104

Contributed by Stanley M. Gartler, July 23, 1998

ABSTRACT G2 was defined originally as the temporal cell cycle (9). We address two questions. Does this very late gap between the termination of DNA replication and the replication occur during what is usually termed G2? What beginning of mitosis. In human cells, the G2 period was proportion of the genome replicates in this late compartment? estimated to be 3–4 h. However, the absence of replicative DNA synthesis during this period designated G2 has never been shown conclusively. In this report, we show that, at MATERIALS AND METHODS some autosomal and X linked loci, programmed DNA rep- Cell Culture. Lymphoblastoid cell lines were grown as lication continues within 90 min of mitosis. Furthermore, described (9). Cell lines used in this study included: FF, derived the major accumulation of cyclin B1, a cell-cycle marker that from a normal male (5); VK and SK, derived from normal is usually ascribed to G2, overlaps extensively with very late females; and 5106, derived from a phenotypically normal male DNA replication. We conclude that the G2 period is much (kindly provided by B. A. Oostra, Erasmus University, Rot- shorter than previously thought and may, in some cells, be terdam, Netherlands, and described below). Fresh blood lym- nonexistent. phocytes were taken from two normal females, isolated by centrifugation over a Ficoll gradient, and cultured at 5 ϫ 106 The eukaryotic cell cycle consists of an orderly series of events cells per ml in RPMI medium 1640 plus 10% fetal bovine in which cells grow, replicate their DNA, and segregate the serum. Cells were stimulated to divide with the addition of 20 duplicated genome to the daughter cells. Our current textbook ␮l͞ml phytohemagglutanin for 3 days before BrdUrd labeling. view of the cell cycle was first put forth by Howard and Pelc General PCR Conditions. All PCRs contained 18.2 ␮lof (1) as encompassing four phases: S phase, the time in which PCR Supermix (GIBCO͞BRL), each primer at 2 ␮M, and 1 ␮l replicative DNA synthesis occurs; M phase, the time in which of the BrdUrd-labeled DNA isolated from each fraction. mitotic chromosome segregation and cell division proceed; Parameters for all PCRs included denaturation at 95°C for 5 and two gap phases, G1 and G2, that temporally separate S min and 23 cycles that included denaturation at 95°C for 1 min, phase from mitosis. annealing at a temperature described below for each primer The G2 phase was inferred from the length of time between pair for 2 min, and extension at 72°C for 3 min. A final the addition of radiolabeled DNA precursors and the appear- extension was carried out at 72°C for 7 min. PCR products were ance of labeled, condensed mitotic chromosomes (1). Auto- run on 1.2% Tris-base, acetic acid, EDTA (TAE) agarose gels. radiography, however, may not be sufficiently sensitive to Transfer of DNA to Hybond Nϩ membrane filters was carried detect low levels of replicative synthesis in the G2 phase. The out with 0.4 M NaOH, followed by neutralization with 0.4 M absence of detectable incorporation of radiolabel could thus Na2HPO4. Hybridization was carried out at 65°C in FBI be taken to indicate erroneously the absence of DNA repli- solution (10% polyethylene glycol 8000͞7% SDS͞1.5ϫ stan- cation. Furthermore, DNA repair synthesis can occur through- dard saline phosphate͞EDTA͞100 ␮g͞ml sheared salmon out the cell cycle (2, 3); replicative and repair DNA synthesis sperm DNA) for at least 3 h. Thereafter, filters were washed are not easily distinguishable by the autoradiographic method. twice in 3ϫ standard saline citrate with 1% SDS, then washed An additional problem is that no cellular marker signaling the twice in 1ϫ standard saline citrate with 1% SDS, and exposed termination of replicative DNA synthesis has been identified. to x-ray film. These features contribute to an inherent lack of both sensitivity The following annealing temperatures were used: G6PD at and specificity for accurately defining the terminal border of 50°C, F9 at 60°C, PGK1 at 62°C, FMR1 at 60°C, inter-Alu 6–84 S phase. at 45°C, inter-Alu 6–82 at 45°C, inter-Alu H-1 at 40°C, and Our studies on fragile sites and the replication timing of inter-Alu H-8 at 40°C. The oligonucleotide primer pairs used FMR1, the gene responsible for the fragile X syndrome, first for PCR of G6PD, F9, and PGK1 have been described (5). The brought our attention to the possibility of replication very close primer pair and corresponding size used for FMR1 are as to mitosis (4). In cell lines derived from patients with fragile follows: fmr69 (5Ј-gagtcaagtggggaagactaagttgc-3Ј) and fmr357 X syndrome, the replication of a large region surrounding the (5Ј-gaactttgtgcataccctctgcttcc-3Ј) (291 bp). The oligonucleo- ͞ FMR1 gene is abnormally late, occurring within the G2 M tide primer pairs employed for allele-specific inter-Alu PCR compartment defined by DNA content (5, 6). Surprisingly, and the corresponding sizes are as follows: inter-Alu 6–84, equally late replication was observed for several loci on the 6–84F (5Ј-agtatccacaggtatctga-3Ј) and 6–84R (5Ј-ttagaaagta- inactive X chromosome in normal female fibroblasts (7, 8), as cattaggcac-3Ј) (240 bp); inter-Alu 6–82, 6–82A (5Ј- well as on the inactive X chromosome in human–hamster cgatatgaagaaaataaaag-3Ј) and 6–82B (5Ј-atctttaatctcatctccat- hybrids (8). In this report, we have determined more precisely 3Ј) (246 bp); inter-Alu H-1, H-1F (5Јatcccatacatagacca-3Ј) and the timing of late DNA replication in the human cell cycle, by H-1R (5Ј-agtaaagcattgatcca-3Ј) (262 bp); inter-Alu H-8, H-8F using a new, sensitive method for fractionating cells late in the (5Ј-tagcatttgattttcta-3Ј) and H-8R (5Ј-ttctctattattacgga-3Ј) (225 bp). The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked ‘‘advertisement’’ in †To whom reprint requests should be addressed at: Fred Hutchinson accordance with 18 U.S.C. §1734 solely to indicate this fact. Cancer Research Center, 1100 Fairview Avenue North C3-168, P.O. © 1998 by The National Academy of Sciences 0027-8424͞98͞9511246-5$2.00͞0 Box 19024, Seattle, WA 98109-1024. e-mail: [email protected]. PNAS is available online at www.pnas.org. edu and [email protected].

11246 Cell Biology: Widrow et al. Proc. Natl. Acad. Sci. USA 95 (1998) 11247

Replication Timing with Cyclin B1 Flow Cytometry. Flow electrophoretically on a 1.3% TAE agarose gel and transferred cytometry was performed as described (9), with a few excep- to a Hybond Nϩ membrane. Incorporation of radiolabeled tions. DNA content was measured by resuspending the cells in nucleotides into the PCR products amplified with BrdUrd 1 ml of the staining solution as described (5). In some DNA from each replication-timing fraction was quantified by experiments, 20 ␮l of antibody (FITC-conjugated Cyclin B1 phosphorimager analysis. The percentage of inter-Alu repli- Antibody Reagent Set; PharMingen) was used instead of 0.5 cation ongoing in each cell-cycle fraction was calculated as ␮g of the antibody with no discernible differences in results. described above, yielding percentages of inter-Alu replication Previously, we have described a method for studying repli- of 1.3, 29, 33, 18, 9.8, 4.8, 2.9, 0.8 for G1 through high cyclin B1, cation timing that uses the incorporation of BrdUrd (5). We respectively. used this method with the following changes: the immunopre- The cell line 5106 (used to obtain the data shown in Fig. 3B) cipitation of BrdUrd-labeled DNA was carried out with 1 ␮g was derived from a normal male with a large expansion of the of anti-BrdUrd monoclonal antibody and 16.5 ␮g of rabbit IgG CGG repeat in the FMR1 gene but without associated meth- directed against mouse IgG; lyophilized BrdUrd DNA isolated ylation (kindly provided by B. A. Oostra). We did not detect from each sorting fraction was dissolved in a volume of any differences in the replication-timing profiles of several Tris-EDTA to obtain the equivalent of 400 cells per ␮l; and specific inter-Alu sequences between this and other cell lines, BrdUrd-labeled CHO-YH21 DNA was not added. All cell confirming that the expansion of the CGG repeat does not fractionation isolates were controlled internally by assaying have a general effect on replication timing (5). In addition, characteristic early and late replicating sequences (for exam- comparable results were obtained by using the normal male ple, see H-8, Fig. 3C). Individual sequence profiles were cell line, FF. obtained in at least two different cell lines. Quantitation of the hybridization signals was performed by densitometry analysis. To calculate the percent of DNA rep- RESULTS lication in the mid- and high cyclin B1 fractions for loci on the Cell-Cycle Fractionation for Replication Timing. To inves- inactive X chromosome, we assumed that the total signal tigate very late DNA replication, we have developed a method constituted the later replication signal. In profiles for which using cyclin B1 to identify cells at different times within the late both phosphorimager analysis and densitometry data were periods of the cell cycle (9). Levels of this cell-cycle protein rise available, the calculated percent of total DNA replication was abruptly as cells obtain a 4C DNA content, peak during the highly comparable. metaphase͞anaphase transition, and fall precipitously as cells Isolation and Analysis of Autosomal Late Replicating Se- complete mitosis (11). The increased level of cyclin B1 is thus quences. We isolated a number of sequences by PCR ampli- a useful marker for cells very late in the cell cycle; this increase fication between closely spaced Alu repeats by using the in cyclin B1 has been considered a marker for G2 and the early primer Alu 3.2 (5Ј-gcgacagagcgagactccgtctc-3Ј). This primer is stages of mitosis (12, 13). Our flow cytometry method takes specific to the 3Ј end of the Alu consensus sequences that advantage of this accumulation of cyclin B1 to sort cell represent five of the eight currently defined subclasses (10). populations progressively closer to the mitotic transition. We ͞ BrdUrd DNA isolated from either the G2 M cell-cycle fraction analyzed eight cell-cycle fractions isolated on this basis: the G1 or the high cyclin B1 fraction was used to amplify late phase fraction, four S phase fractions that were clearly be- replicating inter-Alu sequences. PCR conditions were as fol- tween the 2C and 4C peaks (S1–S4) and three fractions with lows: denaturation at 95°C for 5 min; 30 cycles that included a DNA content of about 4C with low, mid-, and high levels of denaturation at 95°C for 1 min, 65°C for 2 min, and 76°C for cyclin B1 (Fig. 1). We previously estimated that 4C cells with 3 min; and a final extension of 76°C for 5 min. PCR products were then cloned using the TA Cloning Kit (Invitrogen). Individual clones that were confirmed as replicating late in the cell cycle were then used for further analysis. The chromo- somal location of each sequence was determined either by probing inter-Alu products amplified from a human-rodent somatic cell mapping panel (Coriell Cell Repositories, Cam- den, NJ) or by directly performing the locus-specific PCR on the same hybrid DNA. Inter-Alu clones 6–84, 6–82, H-1, and H-8 are located on chromosomes 4, 12, 10, and X, respectively. Quantification of Generalized Inter-Alu PCR. One micro- liter of BrdUrd DNA equal to 400 cell equivalents from each fraction was amplified by using the Alu3.2 primer as described above, except that the products were amplified with 28 cycles. Then, the products were separated electrophoretically and transferred to a Hybond Nϩ membrane. The blot was then hybridized at 42°C overnight in FBI hybridization solution. The blot was washed with 6ϫ standard saline citrate and 1% SDS at 42°C for 60 min and either exposed to x-ray film or quantified by phosphorimager analysis. The percentage of inter-Alu replication detected in each fraction was determined by dividing the measured signal intensity in each lane by the total measured intensity. An alternative quantitative approach incorporated radio- nucleoside triphosphates. PCR was performed with 200 ␮M dGTP, 200 ␮M dATP, 100 ␮M dCTP, 100 ␮M TTP, 10 ␮Ci FIG. 1. Cell-cycle fractionation by cyclin B1 flow cytometry. Human lymphoblastoid cells were labeled with BrdUrd for 90 min (5), of [32P]dCTP, 10 ␮Ci [32P]TTP, 4 ␮M Alu3.2, 1.25 units Taq ␮ ␮ fixed, and separated on the basis of DNA content and cyclin B1 levels. polymerase, and 1 l of BrdUrd DNA in a 20- l reaction by The cyclin B1 units are arbitrary. Equal numbers of cells (between using a standard buffer (Boehringer Mannheim). The inter- 5,000 and 10,000) were collected from each sorting fraction (G1; Alu PCR was run at the conditions described above, except S1–S4; and low, mid-, and high cyclin B1) and used for subsequent that 23 cycles were performed. Products were separated analysis of replication timing (modified from ref. 9). 11248 Cell Biology: Widrow et al. Proc. Natl. Acad. Sci. USA 95 (1998) low and mid-levels of cyclin B1 transit to high levels of cyclin occurred on average within 90 min of mitosis. More generally, B1 in about 3.0 and 1.6 h, respectively (9). the average sum of the inactive X replication signals in the mid- Replication Timing of X Linked Loci. We coupled our and high cyclin B1 fractions is 71% (range: 48–84%); thus, cyclin B1 method of fractionating cells and our 5Ј bromode- over two-thirds of the cells have replicated these loci within 3 h oxyuridine (BrdUrd) method to isolate newly replicated DNA of mitosis. (5) to examine the replication timing for four X linked loci; Replication Timing of Autosomal Loci. Does replication of glucose-6-phosphate dehydrogenase (G6PD; Xq28), blood fac- sequences other than those on the inactive X chromosome also tor IX (F9; Xq26.3), fragile X mental retardation 1 (FMR1; extend into this very late part of the cell cycle? We charac- Xq27.3), and phosphoglycerate kinase 1 (PGK1; Xq13.3). In terized the replication timing of several autosomal loci that normal male lymphoblasts, the X linked loci analyzed have were selected for replicating late in the cell cycle (see Materials profiles representative of the replication timing for these and Methods). Profiles from four late replicating autosomal regions on an active X chromosome (Fig. 2A). In normal loci show that replication had occurred in cells subsequently female lymphoblasts, a bimodal replication pattern is seen for collected in all three cyclin B1 fractions, including the high each locus, with the later replication signal representing the cyclin B1 fraction (Fig. 3A; data not shown for the two replication timing of the locus on the inactive X chromosome additional loci). The high cyclin B1 fraction contains on (7, 8). The high cyclin B1 fraction contains on average 24% average 13% (range: 7–19%) of the replication signal for these (range: 18–31%) of the replication signal for loci on the autosomal sequences. This result indicates that the DNA inactive X chromosome. The frequent detection of a locus in cells recovered from the high cyclin B1 fraction indicates that the replication of this sequence commonly overlaps the major increase in cyclin B1. In addition, we have observed previously that about 50% of the cells in the high cyclin B1 fraction are mitotic (9). Given this observation, and the 90 min BrdUrd- labeling period, we estimate that DNA replication detected in cells recovered from the high cyclin B1 fraction must have

FIG. 3. Detection of very late DNA replication for autosomal loci. (A) Replication of two inter-Alu sequences located on chromosomes 4 and 12 is highly restricted and very late in the cell cycle in lymphoblasts. The high cyclin B1 fraction contains on average 13% of the replication signal for these loci. (B) In phytohemagglutinin- FIG. 2. Detection of very late DNA replication for loci on the stimulated lymphocytes, replication of the same autosomal inter-Alu inactive X chromosome. (A) Newly replicated DNA at four loci on the sequences described in A is also highly restricted and very late in the inactive X chromosome is detected in lymphoblasts recovered from all cell cycle. In this case, the high cyclin B1 fraction contains 26% of the three cyclin B1 fractions. Replication-timing profiles for several X replication signal for these autosomal sequences. (C) Replication linked loci in male and female cell lines are shown. The high cyclin B1 timing for a heterogeneous population of inter-Alu sequences is spread fraction contains on average 24% of the replication signal for loci on throughout the cell cycle in lymphoblasts, with about 1% occurring the inactive X chromosome. (B) In phytohemagglutinin-stimulated within the high cyclin B1 fraction. The percentage of the total lymphocytes, newly replicated DNA at four loci on the inactive X inter-Alu replication detected by phosphorimager analysis is shown for chromosome is detected in cells recovered from all three cyclin B1 each cell-cycle fraction. The same membrane was stripped of the fractions. The high cyclin B1 fraction contains on average 22% of the labeled primer and reprobed with a specific late replicating inter-Alu replication signal for loci on the inactive X chromosome. The detection sequence found on the X chromosome (H-8); the results demonstrated of newly replicated DNA in the high cyclin B1 fraction demonstrates the specificity of this approach. An alternative quantification method that the replication of sequences close to mitosis in normal human cells in which radioactive deoxyribonucleoside triphosphates were incor- is not a consequence of cell transformation. porated during the PCR gave similar results. Cell Biology: Widrow et al. Proc. Natl. Acad. Sci. USA 95 (1998) 11249 replication of several autosomal sequences overlaps with the because no additional major signal is seen earlier in the cell accumulation of cyclin B1 in some cells, similar to loci exam- cycle (Fig. 3A and 3B). In addition, we detect a second, later ined on the inactive X chromosome. Furthermore, the detec- component of DNA replication of X linked loci only in female tion of replication in cells recovered from the high cyclin B1 cells (Fig. 2A). The absence of these later replication signals fraction indicates that in some cells autosomal loci replicate for X linked loci in male cells indicates that very late replica- within 90 min of entry into mitosis and during the major tion detected in female cells for these loci is not due to specific increase in cyclin B1. Furthermore, the average sum of the repair replication. replication signals in the mid- and high cyclin B1 fractions is Originally, the length of G2 was estimated autoradiographi- 56% (range: 42–80%), indicating that over half of the cells cally by determining the time at which 50% of metaphases were have replicated these autosomal sequences within3hof labeled after the addition of radioactive DNA precursors (18). mitosis. As expected for most autosomal loci, we did not detect Examination of the data in many of these early publications a significant difference in the replication profiles between the (15–17, 19) reveals that a small percentage of labeled met- male and female cell lines. The phenomenon of very late aphases was seen commonly after 1- to 2-h periods of labeling, replication is therefore not restricted to loci on the inactive X a considerably shorter time than the 3–4 h estimated for G2. chromosome. These data indicate that the length of G2 in some cells may be Generalized Replication Timing. What fraction of total markedly shorter than previously estimated. genomic replication can we assign to this very late replicating Technical limitations of previous methods made the analysis component? We examined the replication timing of a large of late replicating sequences difficult. In addition, the realiza- subset of genomic sequences by using a primer designed to tion that repair synthesis can occur throughout the cell cycle (2, amplify sequences found between closely spaced Alu se- 3), most notably in G , later provided a possible biological quences; we refer to these as inter-Alu sequences. Limited- 2 cycle PCR was used to amplify a heterogeneous population of explanation for the observed G2 labeling, and perhaps dis- inter-Alu sequences found within each replication-timing frac- tracted attention from the possibility of bona fide replicative tion. Quantification of the PCR products provides an estimate synthesis late in the cell cycle. The discovery of and emphasis of the amount of inter-Alu replication that was ongoing in each on cell-cycle checkpoints bolstered the concept of a discrete G2 cell-cycle fraction. Inter-Alu replication is skewed toward the phase and its role as a important compartment in the cell cycle. first half of S phase (Fig. 3C), in agreement with previous The concept of the cell cycle consequently evolved to include studies (14). About 5%, 3%, and 1% of the total inter-Alu DNA replication are detected in cell fractions having low, mid-, and high cyclin B1 levels, respectively. The detectable DNA replication for these sequences in the high cyclin B1 fraction suggests that an appreciable fraction of the genome— about 1%—is replicated on average within 90 min of mitosis; the sum of the replication detected in the mid- and high cyclin B1 fractions indicates that at least 4% replicate within3hof mitosis. Given the overrepresentation of the inter-Alu repli- cation in early S phase, these values for very late DNA replication may be underestimated. Replication Timing in Nontransformed Cells. To determine whether DNA replication close to mitosis also occurs in cells that had not been transformed, we studied the replication timing of several loci in phytohemagglutinin-stimulated lym- phocytes. In such cells, G2 has been estimated to be 3–3.5 h (15–17). Very late replication, detectable in the high cyclin B1 fraction, was observed for both X linked (Fig. 2B) and autosomal sequences (Fig. 3B). The high cyclin B1 fraction contains on average 22% (range: 6–33%) and 26% (range: 20–32%) of the replication signal for inactive X linked and autosomal sequences, respectively. Replication of sequences very late in the cell cycle, overlapping extensively with the major accumulation of cyclin B1, can therefore occur in normal human cells and is not a consequence of cell transfor- mation.

DISCUSSION We have discovered that DNA replication overlaps extensively with the major increase in cyclin B1, previously described as a molecular marker for G2 and the early stages of mitosis (12, 13). Very late DNA replication continues within 90 min of mitosis in some cells, includes loci on the inactive X chromo- some in female cells as well as autosomal loci in both female and male cells, and accounts for at least 1% of the total DNA. We have observed this phenomenon in both fresh blood FIG. 4. The cell cycle. (A) The textbook view: the cell cycle is lymphocytes stimulated with phytohemagglutinin and in lym- divided into four phases; G1, S phase, G2, and mitosis. (B) A more recent view: the accumulation of cyclin B1 identifies a cell as residing phoblasts transformed with Epstein–Barr Virus. in G2 or early mitosis. (C) Our proposed revision: low levels of We conclude that our observations reflect true replicative replicative DNA synthesis (shading) are ongoing much later in the cell synthesis late in the cell cycle rather than repair synthesis. For cycle than previously suspected, overlapping extensively with the a particular autosomal late replicating sequence, we can assign accumulation of cyclin B1. In some cells, the existence of a discrete G2 at least part of the detected signal to replicative DNA synthesis, phase is questioned. 11250 Cell Biology: Widrow et al. Proc. Natl. Acad. Sci. USA 95 (1998) a discrete G2 phase defined as the period absent of DNA ally, the timing of the replication of such sequences may serve replication before mitosis (Fig. 4A) (20–23), even though early as a cellular signal for entry into mitosis. studies suggested that some DNA replication might occur very late in the cell cycle. The findings presented in this paper We thank Andrew Berger and Michelle Black for excellent assis- demonstrate that programmed replication of specific se- tance with the flow cytometry; Theresa Canfield and Kevin Henne for quences can account for this very late replication. We have technical assistance; Tina Kajimura and Jennifer Stoeck for important defined more precisely the timing of this very late replication, contributions to this work; Brian Reid and members of the Laird Lab for helpful discussions; and Lester Goldstein, Lee Hartwell, Eric Foss, identified some of the sequences involved, and demonstrated and Peter Rabinovich for helpful comments on the manuscript. This that a substantial fraction of cells are experiencing very late work was supported by grants from the National Institutes of Health DNA replication. (GM53805, P30CA1570422, GM07266, GM52463, and HD16659). 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